Lipid-Coated Albumin Nanoparticle Compositions and Methods of Making and Method of Using the Same

ABSTRACT

Lipid nanoparticle formulations, methods of making, and methods of using same are disclosed.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application61/650,729, filed May 23, 2012, and U.S. Provisional Application61/784,892, filed Mar. 14, 2013, the disclosures of which are herebyincorporated by reference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant Numbers R01CA135243, DK088076, and CA152969 awarded by the National Institutes ofHealth. The government has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-web and is hereby incorporated by reference in itsentirety. The ASCII copy, created on May 22, 2013, is named604_(—)55043_SEQ_LIST_OSU-2013-246(2).txt, and is 1,476 bytes in size.

TECHNICAL FIELD

The present disclosure pertains to lipid nanoparticles (LNs) usable forthe delivery of therapeutic compositions, including, but not limited tonucleic acids (NAs).

BACKGROUND OF THE INVENTION

A liposome is a vesicle composed of one or more lipid bilayers, capableof carrying hydrophilic molecules within an aqueous core or hydrophobicmolecules within its lipid bilayer(s). As used herein, “Lipidnanoparticles” (LNs) is a general term to described lipid-basedparticles in the submicron range. LNs can have structuralcharacteristics of liposomes and/or have alternative non-bilayer typesof structures. Drug delivery by LNs via systemic route requiresovercoming several physiological barriers. The reticuloendothelialsystem (RES) is responsible for clearance of LNs from the circulation.Once escaping the vasculature and reaching the target cell, LNs aretypically taken up by endocytosis and must release the drug into thecytoplasm prior to degradation within acidic endosome conditions.

In particular, the delivery of such nucleic acids (NAs), including siRNAand other therapeutic oligonucleotides is a major technical challengethat has limited their potential for clinical translation.

The development of efficient delivery vehicles is a key to clinicaltranslation of oligonucleotide (ON) therapeutics. It is desired that aLN formulation should be able to (1) protect the drug from enzymaticdegradation; (2) traverse the capillary endothelium; (3) specificallyreach the target cell type without causing excessive immunoactivation oroff-target cytotoxicity; (4) promote endocytosis and endosomal release;and (5) form a stable formulation with high colloidal stability and longshelf-life.

SUMMARY OF THE INVENTION

Provided herein are LNs that encapsulate therapeutic oligonucleotideswith high efficiency and fulfill physical and biological criteria forefficacious delivery. In certain embodiments, the LNs comprisehyper-cationized and/or pH-responsive HSA-polymer conjugates. In certainembodiments, the HSA-polymer conjugate comprises HSA-PEI or HSA-PEHA.The incorporation of hyper-cationized albumin-polymer conjugates (APC)increases the transfection efficiency of LN formulations. These LNsparticles are also described herein as lipid-coated albuminnanoparticles (LCANs)

In a first aspect, provided herein is a lipid nanoparticle (LN)comprising at least one lipid and albumin conjugated to a positivelycharged polymer.

In certain embodiments, the LN comprises a hyper-cationizedalbumin-polycation conjugate (APC). In certain embodiments, thepolycation comprises a polyamine selected from the group consisting ofspermine, dispermine, trispermine, tetraspermine, oligospermine,thermine, spermidine, dispermidine, trispermidine, oligospermidine,putrescine, polylysine, polyarginine, a polyethylenimine of branched orlinear type, and polyallylamine.

In certain embodiments, the positively-charged polymer consistsessentially of a polyethylenimine.

In certain embodiments, the polyethylenimine has a molecular weight notgreater than 50 kDa, or from about 200 Da to about 2000 Da. In certainembodiments, the positively-charged polymer comprisespentaethylenehexamine (PEHA) or tetraethylenepentamine (TEPA).

In certain embodiments, the LN comprises a polyethylenimine conjugatedto human serum albumin.

In certain embodiments, the conjugation is via one or more cross linkingagents. In certain embodiments, the LN comprises PEHA conjugated to HSA.

In certain embodiments, multiple PEHA molecules are linked to each HSAmolecule. For example, in certain embodiments, between about two (2) andabout twenty (2) PEHA molecules can be linked to each HSA molecule. Incertain embodiments, eleven (11) PEHA molecules are linked to each HSAmolecule.

In certain embodiments, the LN comprises a mixture of two or more lowmolecular weight polymers.

In certain embodiments, the at least one lipid comprises a cationiclipid, a neutral lipid, and a PEGylated lipid, with or withoutcholesterol.

In certain embodiments, at least one lipid comprises1,2-dioleoyl-3-trimethylammonium-propane (DOTAP),L-α-phosphatidylcholine (SPC), and d-alpha-tocopheryl polyethyleneglycol 1000 succinate (TPGS). In particular embodiments, the lipids arein a 25:70:5 molar ratio of DOTAP:SPC:TPGS.

In certain embodiments, the LN encapsulates molecules selected fromnucleic acids, chemo therapeutic agents, or combinations thereof. Incertain embodiments, the encapsulated molecules comprise a nucleic acidselected from plasmid DNAs, antisense oligonucleotides, miRs, anti-miRs,shRNAs, siRNAs, or combinations thereof. In certain embodiments, theencapsulation rate of therapeutic agents or nucleotides is 40% orhigher.

In certain embodiments, the LN has a diameter under 300 nm, or under 200nm, or about 98 nm.

In certain embodiments, the polymer is bound only to an external surfaceof the nanoparticle via direct connection or via a crosslinker.

In certain embodiments, the LN further comprises a polyethyleneglycol-conjugated lipid. In certain embodiments, the polyethyleneglycol-conjugated lipid is selected from the group consisting ofpolysorbate 80, TPGS, and mPEG-DSPE. In particular embodiments, thepolyethylene glycol-conjugated lipid is present at a concentration lessthan about 15.0 molar percent.

In certain embodiments, the LN further comprises a ligand capable ofbinding to a target cell or a target molecule. In certain embodiments,the ligand is an antibody or an antibody fragment. In certainembodiments, the ligand is selected from cRGD, galatose-containingmoieties, transferrin, folate, low density lipoprotein, or epidermalgrowth factors.

In another broad aspect, provided herein is a pharmaceutical compositioncomprising a lipid nanoparticle having at least one lipid and albuminconjugated to a positively charged polymer, and a pharmaceuticallyacceptable excipient.

In certain embodiments, the pharmaceutical composition is administeredperorally, intravenously, intraperitoneally, subcutaneously, ortransdermally. In particular embodiments, the pharmaceutical compositionis prepared as an orally administered tablet, a sterile solution, asterile suspension, a lyophilized powder, or a suppository.

In another broad aspect, provided herein is a method of making alipid-coated albumin nanoparticle (LCAN). The method involvessynthesizing a human serum albumin-pentaethylenehexamine (HSA-PEHA)conjugate; adding at least one lipid to the HSA-PEHA conjugate; adding anucleic acid to the mixture of lipids and the HSA-PEHA conjugate toobtain an LCAN precursor; and subjecting the LCAN precursor to adialysis or diafiltration step to make a lipid-coated albuminnanoparticle.

In certain embodiments, the at least one lipid comprises DOTAP, SPC, andTPGS at a 25:70:5 ratio. In certain embodiments, the nucleic acid isselected from pDNAs, antisense oligonucleotides, miRs, anti-miRs,shRNAs, siRNAs, or combinations thereof. Further provided herein is theproduct made from the described method.

In another broad aspect, provided herein is a method of diagnosing ortreating a cancer or infectious disease. The method involvesadministering an effective amount of a pharmaceutical compositioncomprising at least one lipid, albumin conjugated to apositively-charged polymer, and a pharmaceutically acceptable excipient,to a patient in need thereof.

In another broad aspect, provided herein is a delivery system comprisingat least one lipid and a macromolecule conjugated to a polymer, whereinthe macromolecule forms an electrostatic complex with a nucleic acid.

In another broad aspect, provided herein is a method of using a lipidnanoparticle. The method involves encapsulating a nucleic acid in alipid nanoparticle, wherein the lipid nanoparticle comprises albuminconjugated to a polymer, incorporating the lipid nanoparticle into apharmaceutical composition, and administering the pharmaceuticalcomposition to a patient in need thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Gel mobility shift analysis of HSA-PEI(600)(APC)-oligodeoxynucleotide (ODN) complexes at varyingODN-to-HSA-PEI(600) w/w ratios. LOR-2501, an ASO against ribonucleasereductase R1 (RNR R1) subunit (purchased from Alpha DNA) was used.

FIG. 2: Zeta potential of LN (LN)-HSA-PEI(600)-LOR-2501 (APC-ODN)complexes.

FIGS. 3A-B: Downregulation of RNR R1 mRNA expression by LOR-2501 inLCANs. The LCANs were prepared at varying APC concentrations underdifferent media conditions: FIG. 3A displays serum-free media; FIG. 3Bdisplays media containing 10% FBS. RNR R1 mRNA expression relative toactin was determined by RT-PCR where untreated KB cells served as abaseline for mRNA expression.

FIG. 4: Cell viability study of KB cells treated withLCAN-HSA-PEI(600)-LOR-2501 (APC) complex. Transfection was performed inserum-free media. Cell viabilities are expressed as a percentagerelative to the mean viability of the untreated KB cells.

FIG. 5: Gel mobility shift analysis of HSA-PEHA-LOR-2501 (APC-ODN)complexes at varying ODN-to-APC w/w ratios. LOR-2501 was used as the ODNin this study.

FIG. 6: Downregulation of RNR R1 mRNA expression by LOR-2501 in LCANs.The LCANs were prepared at varying APC concentrations under serum-freeconditions. RNR R1 mRNA expression relative to actin was determined byRT-PCR where untreated KB cells served as a baseline for mRNAexpression.

FIG. 7: Bcl-2 down regulation in KB cells by lipid-coated albuminnanoparticle (LCAN)-G3139 as compared to LN-G3139.

FIG. 8: An example scheme for synthesizing an APC.

FIG. 9: The mechanism of action for hyper-cationized pH-responsive APCs.

FIG. 10: Upregulation of p27/kip1 mRNA by LCAN loaded with anti-miR-221in CAL-51 breast cancer cells.

FIG. 11: Upregulation of estrogen mRNA by LCAN loaded with anti-miR-221in CAL-51 cells. The estrogen receptor is a target of miR-221.

DETAILED DESCRIPTION OF THE INVENTION

Those of ordinary skill in the art will realize that the followingdetailed description of the embodiments is illustrative only and notintended to be in any way limiting. Other embodiments will readilysuggest themselves to such skilled persons having the benefit of thisdisclosure. Reference to an “embodiment,” “aspect,” or “example” hereinindicate that the embodiments of the invention so described may includea particular feature, structure, or characteristic, but not everyembodiment necessarily includes the particular feature, structure, orcharacteristic. Further, repeated use of the phrase “in one embodiment”does not necessarily refer to the same embodiment, although it may.

Not all of the routine features of the implementations or processesdescribed herein are shown and described. It will, of course, beappreciated that in the development of any such actual implementation,numerous implementation-specific decisions will be made in order toachieve the developer's specific goals, such as compliance withapplication- and business-related constraints, and that these specificgoals will vary from one implementation to another and from onedeveloper to another. Moreover, it will be appreciated that such adevelopment effort might be complex and time-consuming, but wouldnevertheless be a routine undertaking for those of ordinary skill in theart having the benefit of this disclosure.

General Description

Nucleic acid (NA)-based therapies are being developed to promote orinhibit gene expression. As mutations in genes and changes in miRNAprofile are believed to be the underlying cause of cancer and otherdiseases, NA-based agents can directly act upon the underlying etiology,maximizing therapeutic potential. Non-limiting examples of NA-basedtherapies include: plasmid DNA (pDNA), small interfering RNA (siRNA),small hairpin RNA (shRNA), microRNA (miR), mimic (mimetic),anti-miR/antagomiR/miR inhibitor, and antisense oligonucleotide (ASO).Until the development of the nanoparticle compositions described herein,the clinical translation of NA-based therapies faced several obstaclesin their implementation since transporting NAs to their intracellulartarget was particularly challenging and since NAs are relativelyunstable and subject to degradation by serum and cellular nucleases.Further, the high negative charges of NAs made it impossible fortransport across the cell membrane, further limiting utility.

The LNs described herein provide a useful platform for the delivery ofboth traditional therapeutic compounds and NA-based therapies. Drugsformulated using LNs provide desirable pharmacokinetic (PK) propertiesin vivo, such as increased blood circulation time and increasedaccumulation at the site of solid tumors due to enhanced permeabilityand retention (EPR) effect. Moreover, in certain embodiments, the LNsmay be surface-coated with polyethylene glycol to reduce opsonization ofLNs by serum proteins and the resulting RES-mediated uptake, and/orcoated with cell-specific ligands to provide targeted drug delivery.

It is desired that the zeta potential of LNs not be excessively positiveor negative for systemic delivery. LNs with a highly positive chargetend to interact non-specifically with non-target cells, tissues, andcirculating plasma proteins, and may cause cytotoxicity. Alternatively,LNs with a highly negative charge cannot effectively incorporate NAs,which are themselves negatively charged, and may trigger rapidRES-mediated clearance, reducing therapeutic efficacy. LNs with aneutral to moderate charge are best suited for in vivo drug and genedelivery.

In certain embodiments, the LNs described herein comprisehyper-cationized albumin-polymer conjugates (APCs). As used herein, theterm “hyper-cationized” mean each polycation carries multiple positivecharges. In particular embodiments, up to 20 polycations can be linkedto each albumin molecule. These factors result in a much higher overallcharge content for APCs compared to traditional cationized albumin,which typically comprises an albumin conjugate where carboxyl groups arereplaced with single-positive-charge amine functional groups. Because oftheir high charge density, the APCs are able to very efficientlyinteract with polyanions such as an oligonucleotide particle, molecule,compound or formulation having multiple cations or positive charges.

The term “lipid nanoparticle” (LP) as used herein refers to a vesicleformed by one or more lipid components. The lipid components describedherein may include cationic lipids. Cationic lipids are lipids thatcarry a net positive charge at any physiological pH. In certainembodiments, the positive charge is used for association with negativelycharged therapeutics such as ASOs via electrostatic interaction.

Suitable cationic lipids include, but are not limited to:3β-[N—(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride(DC-Chol); 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP);1,2-dioleoyl-3-dimethylammonium-propane (DODAP);dimethyldioctadecylammonium bromide salt (DDAB);1,2-dilauroyl-sn-glycero-3-ethylphosphocholine chloride (DL-EPC);N-[1-(2,3-dioleyloxy)propyl]-N—N—N-trimethyl ammonium chloride (DOTMA);N-[1-(2,3-dioleyloxy)propyl]-N—N—N-dimethyl ammonium chloride (DODMA);N,N-dioctadecyl-N,N-dimethylammonium chloride (DODAC);N-(1-(2,3-dioleyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammoniumtrifluoracetate (DOSPA); 1,2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide (DMRIE); dioctadecylamidoglycylspermine (DOGS); neutrallipids conjugated to cationic modifying groups; and combinationsthereof. In addition, a number of cationic lipids in availablepreparations could be used, such as LIPOFECTIN® (from GIBCO/BRL),LIPOFECTAMINE® (from GIBCO/MRL), siPORT NEOFX® (from AppliedBiosystems), T RANSFECTAM® (from Promega), and TRANSFECTIN® (fromBio-Rad Laboratories, Inc.). The skilled practitioner will recognizethat many more cationic lipids are suitable for inclusion in the LNformulations. In certain embodiments, the cationic lipids may be presentat concentrations up to about 80.0 molar percent of total lipids in theformulation, or from about 5.0 to about 50.0 molar percent of theformulation.

In certain embodiments, the LN formulations presently disclosed may alsoinclude anionic lipids. Anionic lipids are lipids that carry a netnegative charge at physiological pH. These anionic lipids, when combinedwith cationic lipids, are useful to reduce the overall surface charge ofLNs and introduce pH-dependent disruption of the LN bilayer structure,facilitating nucleotide release by inducing nonlamellar phases at acidicpH or induce fusion with the cellular membrane.

Examples of suitable anionic lipids include, but are not limited to:fatty acids such as oleic, linoleic, and linolenic acids; cholesterylhemisuccinate;1,2-di-O-tetradecyl-sn-glycero-3-phospho-(1′-rac-glycerol) (diether PG);1,2-dimyristoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (sodium salt);1,2-dimyristoyl-sn-glycero-3-phospho-L-serine (sodium salt);1-hexadecanoyl, 2-(9Z,12Z)-octadecadienoyl-sn-glycero-3-phosphate;1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DOPG);dioleoylphosphatidic acid (DOPA); and1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS); anionic modifyinggroups conjugated to neutral lipids; and combinations thereof. Theanionic lipids of the present disclosure are present at concentrationsup to about 60.0 molar percent of the formulation, or from about 5.0 toabout 25.0 molar percent of the formulation.

In certain embodiments, charged LNs are advantageous for transfection,but off-target effects such as cytotoxicity and RES-mediated uptake mayoccur. Hydrophilic molecules such as polyethylene glycol (PEG) may beconjugated to a lipid anchor and included in the LNs described herein toprevent LN aggregation or interaction with membranes. Hydrophilicpolymers may be covalently bonded to lipid components or conjugatedusing crosslinking agents to functional groups such as amines.

Suitable conjugates of hydrophilic polymers include, but are not limitedto: polyvinyl alcohol (PVA); polysorbate 80;1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-PEG2000(DSPE-PEG2000); D-alpha-tocopheryl polyethylene glycol 1000 succinate(TPGS); dimyristoylphosphatidylethanolamine-PEG2000 (DMPE-PEG2000); anddipalmitoylphosphatidylethanolamine-PEG2000 (DPPE-PEG2000). In certainembodiments, the hydrophilic polymer may be present at concentrationsranging from about 0 to about 15.0 molar percent of the formulation, orfrom about 5.0 to about 10.0 molar percent of the formulation. Also, incertain embodiments, the molecular weight of the PEG used is betweenabout 100 and about 10,000 Da, or from about 100 to about 2,000 Da.

The LNs described herein may further comprise neutral and/or cholesterollipids as helper lipids. These lipids are useful to stabilize theformulation, reduce elimination in vivo, or increase transfectionefficiency. The LNs may be formulated in a solution of saccharides suchas, but not limited to, glucose, sorbitol, sucrose, maltose, trehalose,lactose, cellubiose, raffinose, maltotriose, dextran, or combinationsthereof, to promote lyostability and/or cryostability.

Neutral lipids have zero net charge at physiological pH. One or acombination of several neutral lipids may be included in any LNformulation disclosed herein.

Suitable neutral lipids include, but are not limited to:phosphatidylcholine (PC, e.g., DSPC, DPPC, DOPC, DMPC, soyPC, eggPC,HSPC), phosphatidylethanolamine (PE, e.g., DOPE, DSPE, DPPE, DSPE,DMPE), ceramide, cerebrosides, sphingomyelin, cephalin, cholesterol,diacylglycerol, glycosylated diacylglycerols, prenols, lysosomal PLA2substrates, N-acylglycines, and combinations thereof.

Other suitable lipids include, but are not limited to: phosphatidicacid, (PG, e.g., DSPG, DMPG, DPPG), and lysophosphatidylethanolamine;sterols such as cholesterol, demosterol, sitosterol, zymosterol,diosgenin, lanostenol, stigmasterol, lathosterol, anddehydroepiandrosterone; and sphingolipids such as sphingosines,ceramides, sphingomyelin, gangliosides, glycosphingolipids,phosphosphingolipids, phytoshingosine; and combinations thereof.

The LN formulations described herein may further comprise fusogeniclipids or fusogenic coatings to promote membrane fusion. Examples ofsuitable fusogenic lipids include, but are not limited to, glycerylmono-oleate, oleic acid, palmitoleic acid, phosphatidic acid,phosphoinositol 4,5-bisphosphate (PIP₂), and combinations thereof.

The LN formulations described here may further comprise cationicpolymers or conjugates of cationic polymers. Cationic polymers orconjugates thereof may be used alone or in combination with lipidnanocarriers. Suitable cationic polymers include, but are not limitedto: polyethylenimine (PEI); pentaethylenehexamine (PEHA); spermine;spermidine; poly(L-lysine); poly(amido amine) (PAMAM) dendrimers;polypropyleneiminie dendrimers; poly(2-dimethylamino ethyl)-methacrylate(pDMAEMA); chitosan; tris(2-aminoethyl)amine and its methylatederivatives; and combinations thereof. In certain embodiments, the chainlength and branching are important considerations for the implementationof polymeric delivery systems. High molecular weight polymers such asPEI (MW 25,000) are useful as transfection agents, but suffer fromcytotoxicity. Low molecular weight PEI (MW 600) does not causecytotoxicity, but is limited due to its inability to facilitate stablecondensation with NAs. As described herein the conjugation of lowmolecular weight polymers to a larger molecule such as albumin is a thusa useful method of increasing activity of electrostatic complexationwith NA condensation while lowing cytotoxicity of LN formulations.

Anionic polymers may be incorporated into the LN formulations presentlydisclosed as well. Suitable anionic polymers include, but are notlimited to: poly(propylacrylic acid) (PPAA); poly(glutamic acid) (PGA);alginates; dextran derivatives; xanthans; derivatized polymers; andcombinations thereof.

In certain embodiments, the LN formulation includes conjugates ofpolymers. The conjugates may be crosslinked to targeting agents,lipophilic moieties, proteins, or other molecules that increase theoverall therapeutic efficacy. Suitable crosslinking agents include, butare not limited to: N-succinimidyl 3-[2-pyridyldithio]-propionate(SPDP); dimethyl 3,3′-dithiobispropionimidate (DTBP);dicyclohexylcarbodiimide (DCC); diisopropyl carbodiimide (DIC);1-ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC);N-hydroxysulfosuccinimide (Sulfo-NHS); N′—N′-carbonyldiimidazole (CDI);N-ethyl-5-phenylisoxazolium-3′sulfonate (Woodward's reagent K); andcombinations thereof.

The addition of targeting agents to the LN provides increased efficacyover passive targeting approaches. Targeting involves incorporation ofspecific targeting moieties such as, but not limited to, ligands orantibodies against cell surface receptors, peptides, lipoproteins,glycoproteins, hormones, vitamins, antibodies, antibody fragments, andconjugates or combinations of these moieties.

In certain embodiments, the maximization of targeting efficiencyincludes the surface coating of the LN with the appropriate targetingmoiety rather than pre-mixing of the targeting ligand with othercomponents, which results in partial encapsulation of the targetingagent, rendering it inaccessible to the cellular target. This methodoptimizes interaction with cell surface receptors.

It is to be understood that targeting agents may be either directlyincorporated into the LN during synthesis or added in a subsequent step.Functional groups on the targeting moiety as well as specifications ofthe therapeutic application (e.g., degradable linkage) dictate theappropriate means of incorporation into the LN. For example, targetingmoieties that do not have lipophilic regions cannot insert into thelipid bilayer of the LN directly and require prior conjugation to alipid anchor before insertion or must form an electrostatic complex withthe LNs.

Also, under certain circumstances, a targeting ligand cannot directlyconnect to a lipophilic anchor. In these circumstances, a molecularbridge in the form of a crosslinking agent may be utilized to facilitatethe interaction. In certain embodiments, it is advantageous to use acrosslinking agent if steric restrictions of the anchored targetingmoiety prevent sufficient interaction with the intended physiologicaltarget. Additionally, if the targeting moiety is only functional undercertain orientations (e.g., monoclonal antibody), linking to a lipidanchor via crosslinking agent is beneficial. In certain embodiments,other methods of bioconjugation may be used to link targeting agents toLNs. Reducible or hydrolysable linkages may be applied to preventaccumulation of the formulation in vivo and the related cytotoxicity.

Various methods of LN preparation are suitable to synthesize the LNs ofthe present disclosure. For example, ethanol dilution, freeze-thaw, thinfilm hydration, sonication, extrusion, high pressure homogenization,detergent dialysis, microfluidization, tangential flow diafiltration,sterile filtration, and/or lyophilization may be utilized. Additionally,several methods may be employed to decrease the size of the LNs. Forexample, homogenization may be conducted on any devices suitable forlipid homogenization such as an Avestin Emulsiflex C5®. Extrusion may beconducted on a Lipex Biomembrane extruder using a polycarbonate membraneof appropriate pore size (0.05 to 0.2 μm). Multiple particle sizereduction cycles may be conducted to minimize size variation within thesample. The resultant LNs may then be passed through a size exclusioncolumn such as Sepharose CL4B or processed by tangential flowdiafiltration to purify the LNs.

Any embodiment of the LNs described herein may further include ethanolin the preparation process. The incorporation of about 30-50% ethanol inLN formulations destabilizes the lipid bilayer and promoteselectrostatic interactions among charged moieties such as cationiclipids with anionic ASO and siRNA. LNs prepared in high ethanol solutionare diluted before administration. Alternatively, ethanol may be removedby dialysis or diafiltration, which also removes non-encapsulated NA.

In certain embodiment, it is desirable that the LNs be sterilized. Thismay be achieved by passing of the LNs through a 0.2 or 0.22 μm sterilefilter with or without pre-filtration.

Physical characterization of the LNs can be carried through manymethods. For example, dynamic light scattering (DLS) or atomic forcemicroscopy (AFM) can be used to determine the average diameter and itsstandard deviation. In certain embodiments, it is especially desirablethat the LNs have about a 200 nm diameter, or less. Zeta potentialmeasurement via zeta potentiometer is useful in determining the relativestability of particles. Both dynamic light scattering analysis and zetapotential analysis may be conducted with diluted samples in deionizedwater or appropriate buffer solution. Cryogenic transmission electronmicroscopy (Cryo-TEM) and scanning electron microscopy (SEM) may be usedto determine the detailed morphology of LNs.

The LNs described herein are stable under refrigeration for severalmonths. LNs requiring extended periods of time between synthesis andadministration may be lyophilized using standard procedures. Acryoprotectant such as 10% sucrose may be added to the LN suspensionprior to freezing to maintain the integrity of the formulation duringlyophilization. Freeze drying of LN formulations is recommended for longterm stability.

In certain embodiments, the LCANs described herein have a diameter ofless than 300 nm, and, in particular embodiments, between about 50 andabout 200 nm in mean diameter. These LNs show enhanced transfection andreduced cytotoxicity, especially under high serum conditions foundduring systemic administration. The LNs are useful in a wide range ofcurrent therapeutic agents and systems, have high serum stability, andcan be designed for targeted delivery with high transfection efficiency.

Albumin-Polymer Conjugates (APCs)

The utilization of cationic polymers as transfection agents alone and inconjunction with lipids in LNs often benefits transfection efficiency.One polymeric transfection agent is high molecular weightpolyethylenimine (PEI), a large polymer with a molecular weight of ˜25kDa. While PEI25K has been used to deliver pDNA to cells, cytotoxicityhas limited its use in vivo. Less toxic, low molecular weight PEI (MW˜600 kDa) has also been investigated, but this has shown diminishedability to interact with and deliver NAs.

Provided herein are hyper-cationized albumin-polymer conjugates (APCs),which do not have any of the deficiencies of the aforementionedpolymers. APCs may either be used alone to deliver agents such as pDNAor combined with lipid-based formulations to deliver agents such as ASOsand siRNA. Albumin also possesses endosomal lytic activity due to itshydrophobic core, which upon conformational change can be exposed andcan induce bilayer disruption or membrane fusion. In some embodiments,such as the HSA-PEI600 conjugate, the APC has an ionization profile thatis responsive to pH change. The charge density is increased at endosomalpH, which is acidic.

In one embodiment, an APC is combined with a cationic lipid combinationto assemble a cationic lipid-APC-NA nanoparticle, sometimes hereincalled LCAN. In another embodiment, an APC is combined with an anioniclipid combination to assemble a lipid-APC-NA nanoparticle. In certainembodiments, the LNs comprise hyper-cationized APCs. These LNs have hightransfection efficiency without additional cytotoxicity. An examplescheme for synthesizing an APC is shown in FIG. 8. The mechanism ofaction for hyper-cationized pH-responsive APCs is shown in FIG. 9.

Also provided herein are macromolecules conjugated to polymers, such aspositively charged polymers. In one embodiment, a low molecular weightpH-sensitive polymer (polyethylenimine MW 600, PEI600) is conjugated tohuman serum albumin (HSA) via cross linking agents, resulting in ahyper-cationized pH-responsive APC. The addition of HSA-PEI600conjugates to LNs significantly increases downregulation of RRM1 (akaRNR R1) with ASO LOR-2501 (purchased from Alpha DNA) in the presence ofserum without substantial cytotoxicity in KB cells (a subline of HeLa).

In another embodiment, a low molecular weight pentaethylenehexamine(PEHA) is conjugated to HSA via cross linking agents, resulting in ahyper-cationized pH-responsive APC. This particular formulation,referred to as a lipid-coated albumin nanoparticle (LCAN), is especiallyuseful for the delivery of oligonucleotides, such as antisense ODNs,pDNAs, siRNAs, shRNAs, miRs, and anti-miRs. Without wishing to be boundby theory, it is believed HSA-PEHA improves the stability and biologicalactivity of the nanoparticles. In certain embodiments, the lipids inthis formulation are DOTAP, SPC, and TPGS, at a ratio of 25:70:5(mol/mol).

Applications

Depending on the application, the LNs disclosed herein may be designedto favor characteristics such as increased loading of NAs, increasedserum stability, reduced RES-mediated uptake, targeted delivery, or pHsensitive release within the endosome. Because of the varied nature ofLN formulations, any one of the several methods provided herein may beused to achieve a particular therapeutic aim. Cationic lipids, anioniclipids, polyalkenes, neutral lipids, fusogenic lipids, cationicpolymers, anionic polymers, polymer conjugates, peptides, targetingmoieties, and combinations thereof may be utilized to meet specificaims.

The LNs described herein can be used as platforms for therapeuticdelivery of oligonucleotide (ON) therapeutics, such as cDNA, siRNA,shRNA, miRNA, anti-miR, and antisense ODN. These therapeutics are usefulto manage a wide variety of diseases such as various types of cancers,leukemias, viral infections, and other diseases. For instance, targetingmoieties such as cyclic-RGD, folate, transferrin, or antibodies greatlyenhance activity by enabling targeted drug delivery. A number of tumorsoverexpress receptors on their cell surface. Non-limiting examples ofsuitable targeting moieties include transferrin (Tf), folate, lowdensity lipoprotein (LDL), and epidermal growth factors. In addition,tumor vascular endothelium markers such as alpha-v-beta-3 integrin andprostate-specific membrane antigen (PSMA) are valuable as targets forLNs. In certain embodiments, LN formulations having particles measuringabout 300 nm or less in diameter with a zeta potential of less than 50mV and an encapsulation efficiency of greater than 20.0% are useful forNA delivery.

Implementation of embodiments of the LN formulations described hereinalone or in combination with one another synergizes with currentparadigms of LN design.

A wide spectrum of therapeutic agents may be used in conjunction withthe LNs described herein. Non-limiting examples of such therapeuticagents include antineoplastic agents, anti-infective agents, localanesthetics, anti-allergics, antianemics, angiogenesis-inhibitors,beta-adrenergic blockers, calcium channel antagonists, anti-hypertensiveagents, anti-depressants, anti-convulsants, anti-bacterial, anti-fungal,anti-viral, anti-rheumatics, anthelminithics, antiparasitic agents,corticosteroids, hormones, hormone antagonists, immunomodulators,neurotransmitter antagonists, anti-diabetic agents, anti-epileptics,anti-hemmorhagics, anti-hypertonics, antiglaucoma agents,immunomodulatory cytokines, sedatives, chemokines, vitamins, toxins,narcotics, imaging agents, and combinations thereof.

NA-based therapeutic agents are highly applicable to the LN formulationsof the present disclosure. Examples of such NA-based therapeutic agentsinclude, but are not limited to: pDNA, siRNA, miRNA, anti-miRNA, ASO,and combinations thereof. To protect from serum nucleases and tostabilize the therapeutic agent, modifications to the substituent NAbase units and/or phosphodiester linker can be made. Such modificationsinclude, but are not limited to: backbone modifications (e.g.,phosphorothioate linkages); 2′ modifications (e.g., 2′-O-methylsubstituted bases); zwitterionic modifications (6′-aminohexy modifiedODNs); the addition of a terminal lipophilic moiety (e.g., fatty acids,cholesterol, or cholesterol derivatives); and combinations thereof. Themodified sequences synergize with the LN formulations disclosed herein.For example, addition of a 3′-cholesterol to an ODN supplies stabilityto a LN complex by adding lipophilic interaction in a system otherwiseprimarily held together by electrostatic interaction during synthesis.In addition, this lipophilic attachment promotes cell permeation bylocalizing the ODN to the outer leaflet of the cell membrane.

Depending on the therapeutic application, the LNs described herein maybe administered by the following methods: peroral, parenteral,intravenous, intramuscular, subcutaneous, intraperitoneal, transdermal,intratumoral, intraarterial, systemic, or convection-enhanced delivery.In particular embodiments, the LNs are delivered intravenously,intramuscularly, subcutaneously, or intratumorally. Subsequent dosingwith different or similar LNs may use alternative routes ofadministration.

Pharmaceutical compositions of the present disclosure comprise aneffective amount of a LN formulation disclosed herein, and/or additionalagents, dissolved or dispersed in a pharmaceutically acceptable carrier.The phrases “pharmaceutical” or “pharmacologically acceptable” refers tomolecular entities and compositions that produce no adverse, allergic orother untoward reaction when administered to an animal, such as, forexample, a human. The preparation of a pharmaceutical composition thatcontains at least one compound or additional active ingredient will beknown to those of skill in the art in light of the present disclosure,as exemplified by Remington's Pharmaceutical Sciences, 2003,incorporated herein by reference. Moreover, for animal (and human)administration, it will be understood that LN preparations should meetsterility, pyrogenicity, and general safety and purity standards asrequired by FDA Office of Biological Standards.

A composition disclosed herein may comprise different types of carriersdepending on whether it is to be administered in solid, liquid oraerosol form, and whether it need to be sterile for such routes ofadministration as injection. Compositions disclosed herein can beadministered intravenously, intradermally, transdermally, intrathecally,intraarterially, intraperitoneally, intranasally, intravaginally,intrarectally, topically, intramuscularly, subcutaneously, mucosally, inutero, orally, topically, locally, via inhalation (e.g., aerosolinhalation), by injection, by infusion, by continuous infusion, bylocalized perfusion bathing target cells directly, via a catheter, via alavage, in cremes, in lipid compositions (e.g., liposomes), or by othermethod or any combination of the forgoing as would be known to one ofordinary skill in the art (see, for example, Remington's PharmaceuticalSciences, 2003, incorporated herein by reference).

The actual dosage amount of a composition disclosed herein administeredto an animal or human patient can be determined by physical andphysiological factors such as body weight or surface area, severity ofcondition, the type of disease being treated, previous or concurrenttherapeutic interventions, idiopathy of the patient and on the route ofadministration. Depending upon the dosage and the route ofadministration, the number of administrations of a preferred dosageand/or an effective amount may vary according to the response of thesubject. The practitioner responsible for administration will, in anyevent, determine the concentration of active ingredient(s) in acomposition and appropriate dose(s) for the individual subject.

In certain embodiments, pharmaceutical compositions may comprise, forexample, at least about 0.1% of an active compound. Naturally, theamount of active compound(s) in each therapeutically useful compositionmay be prepared is such a way that a suitable dosage will be obtained inany given unit dose of the compound. Factors such as solubility,bioavailability, biological half-life, route of administration, productshelf life, as well as other pharmacological considerations will becontemplated by one skilled in the art of preparing such pharmaceuticalformulations, and as such, a variety of dosages and treatment regimensmay be desirable.

In other non-limiting examples, a dose may also comprise from about 1microgram/kg/body weight, about 5 microgram/kg/body weight, about 10microgram/kg/body weight, about 50 microgram/kg/body weight, about 100microgram/kg/body weight, about 200 microgram/kg/body weight, about 350microgram/kg/body weight, about 500 microgram/kg/body weight, about 1milligram/kg/body weight, about 5 milligram/kg/body weight, about 10milligram/kg/body weight, about 50 milligram/kg/body weight, about 100milligram/kg/body weight, about 200 milligram/kg/body weight, about 350milligram/kg/body weight, about 500 milligram/kg/body weight, to about1000 mg/kg/body weight or more per administration, and any rangederivable therein. In non-limiting examples of a derivable range fromthe numbers listed herein, a range of about 5 mg/kg/body weight to about100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500milligram/kg/body weight of active pharmaceutical ingredient (API),etc., can be administered, based on the numbers described above.

In certain embodiments, a composition herein and/or additional agents isformulated to be administered via an alimentary route. Alimentary routesinclude all possible routes of administration in which the compositionis in direct contact with the alimentary tract. Specifically, thepharmaceutical compositions disclosed herein may be administered orally,buccally, rectally, or sublingually. As such, these compositions may beformulated with an inert diluent or with an assimilable edible carrier.

In further embodiments, a composition described herein may beadministered via a parenteral route. As used herein, the term“parenteral” includes routes that bypass the alimentary tract.Specifically, the pharmaceutical compositions disclosed herein may beadministered, for example but not limited to, intravenously,intradermally, intramuscularly, intraarterially, intrathecally,subcutaneous, or intraperitoneally (U.S. Pat. Nos. 6,753,514, 6,613,308,5,466,468, 5,543,158; 5,641,515; and 5,399,363 are each specificallyincorporated herein by reference in their entirety).

Solutions of the compositions disclosed herein as free bases orpharmacologically acceptable salts may be prepared in water suitablymixed with a surfactant, such as hydroxypropylcellulose. Dispersions mayalso be prepared in glycerol, liquid polyethylene glycols, and mixturesthereof and in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms. The pharmaceutical forms suitable for injectable useinclude sterile aqueous solutions or dispersions and sterile powders forthe extemporaneous preparation of sterile injectable solutions ordispersions (U.S. Pat. No. 5,466,468, specifically incorporated hereinby reference in its entirety). In all cases the form must be sterile andmust be fluid to the extent that easy injectability exists. It must bestable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms, such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (i.e., glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and/or vegetable oils. Proper fluidity may bemaintained, for example, by the use of a coating, such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude agents to achieve isotonicity, for example, sugars or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption such as, for example, aluminum monostearate or gelatin.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous, and intraperitoneal administration. In thisconnection, sterile aqueous media that can be employed will be known tothose of skill in the art in light of the present disclosure. Forexample, one dosage may be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion, (see for example, “Remington's PharmaceuticalSciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variationin dosage will necessarily occur depending on the condition of thesubject being treated. The person responsible for administration will,in any event, determine the appropriate dose for the individual subject.Moreover, for human administration, preparations should meet sterility,pyrogenicity, general safety, and purity standards as required by FDAOffice of Biologics standards.

Sterile injectable solutions are prepared by incorporating thecompositions in the required amount in the appropriate solvent withvarious of the other ingredients enumerated above, as required, followedby sterilization. Generally, dispersions are prepared by incorporatingthe various sterilized compositions into a sterile vehicle whichcontains the basic dispersion medium and the required other ingredientsfrom those enumerated above. In the case of sterile powders for thepreparation of sterile injectable solutions, some methods of preparationare vacuum-drying and freeze-drying techniques which yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof. A powdered composition iscombined with a liquid carrier such as, e.g., water or a salinesolution, with or without a stabilizing agent.

In other embodiments, the compositions may be formulated foradministration via various miscellaneous routes, for example, topical(i.e., transdermal) administration, mucosal administration (intranasal,vaginal, etc.) and/or via inhalation.

In certain embodiments, the compositions may be delivered by eye drops,intranasal sprays, inhalation, and/or other aerosol delivery vehicles.Methods for delivering compositions directly to the lungs via nasalaerosol sprays has been described in U.S. Pat. Nos. 5,756,353 and5,804,212 (each specifically incorporated herein by reference in theirentirety). Likewise, the delivery of drugs using intranasalmicroparticle resins (Takenaga et al., 1998) andlysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725,871,specifically incorporated herein by reference in its entirety) are alsowell-known in the pharmaceutical arts and could be employed to deliverthe compositions described herein. Likewise, transmucosal drug deliveryin the form of a polytetrafluoroetheylene support matrix is described inU.S. Pat. No. 5,780,045 (specifically incorporated herein by referencein its entirety), and could be employed to deliver the compositionsdescribed herein.

It is further envisioned the compositions disclosed herein may bedelivered via an aerosol. The term aerosol refers to a colloidal systemof finely divided solid or liquid particles dispersed in a liquefied orpressurized gas propellant. The typical aerosol for inhalation consistsof a suspension of active ingredients in liquid propellant or a mixtureof liquid propellant and a suitable solvent. Suitable propellantsinclude hydrocarbons and hydrocarbon ethers. Suitable containers willvary according to the pressure requirements of the propellant.Administration of the aerosol will vary according to subject's age,weight and the severity and response of the symptoms.

EXAMPLES

Certain embodiments of the present invention are defined in the Examplesherein. It should be understood that these Examples, while indicatingpreferred embodiments of the invention, are given by way of illustrationonly. From the above discussion and these Examples, one skilled in theart can ascertain the essential characteristics of this invention, andwithout departing from the spirit and scope thereof, can make variouschanges and modifications of the invention to adapt it to various usagesand conditions.

Example 1

HSA (25%) was purchased from Octapharma. PEHA was purchased fromSigma-Aldrich. A stock solution of PEHA, pH adjusted to 8.0 with M HClwas prepared. HSA was combined with 500× of PEHA. Then 80× of the1-ethyl-3-[3dimethylaminopropyl]carbodiimide hydrochloride (EDC) (fromFisher Scientific) was added to the solution under stiffing. Thereaction proceeded at room temperature for >4 h. The produce HSA-PEHAwas purified by gel filtration chromatography on a PD-10 desaltingcolumn or by dialysis using a MWCO 10,000 membrane to remove unreactedPEHA and byproducts. Protein concentration of the product was determinedby a BCA protein assay. The molecular weight of the HSA-PEHA conjugatewas determined by matrix-assisted laser desorption-ionizationtime-of-flight mass spectrometry (MADLI TOF MS). On average, there were11 PEHA linked to each HSA based on the result showing m/z of 66405.756.The product can be stored at 4° C., frozen, or lyophilized.

Example 2

Lipids 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) (Avanti PolarLipids), L-α-phosphatidylcholine derived from soybean (SPC) (AvantiPolar Lipids), and d-alpha-tocopheryl polyethylene glycol 1000 succinate(TPGS) (Eastman Chemical) were dissolved in ethanol. Lipids werecombined at 25:70:5 (mol/mol). Briefly, 3.15, 8.83, and 0.63 μmol ofDOTAP, SPC, and TPGS, respectively, were combined in 4 mL of ethanol.This was then added into 3 mg of HSA-PEHA dissolved in 4 mL of 20 mMHEPES buffer (pH 7.4), followed by 1 mg of an ASO against bcl-2, G3139(purchased from Alpha DNA) in 2 mL of 20 mM HEPES buffer to form amixture containing 40% ethanol. This was then dialyzed against HEPESbuffer using a MWCO 10K Slide-A-Lyzer cassette to remove ethanol andfree G3139. The product, lipid-coated albumin nanoparticle (LCAN)-G3139,was concentrated to 2 mg/mL ODN concentration by diafiltration, 10%sucrose was added into the product, and the product was sterile filteredthrough a 0.2 μm filter. The LCAN-G3139 was stable at 4° C., and wasfrozen or lyophilized for long term storage. The particle size of LCANwas determined by NICOMP 370 particle size analyzer. The zeta potentialwas determined on a zetaPALS instrument. Drug loading efficiency wasdetermined by Oligreen ssDNA quantitation reagents. LCAN particles werefound to be <200 nm in diameter, had a zeta potential of +20˜+40 mV anda G3139 encapsulation efficiency of greater than >60%. The same processwas used to synthesize ligand-conjugated LCANs by incorporatinglipid-derivatized ligands into the lipid components during nanoparticlesynthesis. Possible ligands include transferring, folate, cRGD, orantibodies.

Example 3

HSA-PEHA conjugate was synthesized as follows. HSA (25%) was purchasedfrom Octapharma. PEHA was dissolved in water and the pH was adjusted to8.0 with HCl. A 500-fold excess of PEHA was added to the HSA solution,followed by 80-fold excess of EDC under stiffing. The reaction proceededat room temperature for 4 hr. The product was purified and concentratedby tangential flow diafiltration on a MicroKros cartridge with MW of10,000 against water. The product protein concentration was determinedby BCA protein assay and the PEHA content in the product was determinedby TNBS amine content assay using a PEHA-based standard curve. ThePEHA-HSA ratio was calculated based on surplus in amine content relativeto unmodified HSA and found to be 10.5:1. SDS-PAGE analysis showed thatthe conjugate migrated as a single band, indicating lack ofintermolecular crosslinking in the HSA-PEHA product.

G3139 is an 18-mer phosphorothioate ASO against bcl-2. G3139 purchasedfrom AlphaDNA was dissolved in mM HEPES, pH 7.4. DOTAP/SPC/TPGS at25:70:5 (m/m) was dissolved in ethanol and added to HSA-PEHA diluted in20 mM HEPES, followed by the G3139 solution, resulting in a finalethanol concentration of 40% and ODN:HSA-PEHA:Total lipids ratio of1:3:10 (wt/wt). This resulted in formulation of colloidal complexes,which were precursors to LCAN. This was then diluted 4× by water andsubjected to tangential flow diafiltration on a MicroKros cartridge withMWCO of 30,000 against 5 mM HEPES buffer, pH 7.4. Then, sucrose wasadded to the product (10% final concentration). The LCAN-G3139 was thensterile filtered using a 0.2 μm filter and stored frozen at −20° C. Theproduct was 2 mg/mL in G3139 concentration, determined by OliGreenassay. The percent recovery of G3139 in the product was 67%. Theparticle size of LCAN was analyzed on a NICOMP Particle Sizer Model 370(Particle Sizing Systems, Santa Barbara, Calif.). A volume-weightedGaussian distribution analysis was used to determine the mean particlediameter and size distribution. The zeta potential (4) was determined ona ZetaPALS (Brookhaven Instruments Corp., Worcestershire, N.Y.). Allmeasurements were carried out in triplicate. The particle size was 98±40nm and the zeta potential was +23 mV.

The product LCAN-G3139 was analyzed for bcl-2 down regulation in KB(human carcinoma) cells. KB cells were plated in 6-well plates at adensity of 2×10⁴ cells/cm² 24 h prior to transfection in RPMI 1640 (LifeTechnologies) medium containing 10% FBS and 1% antibiotics. The mediumwas removed and replaced with various G3139 ODN formulations in RPMI1640 culture medium at G3139 concentration of 1 μM. The control LN-G3139is a LN formulation with the composition of DOTAP/SPC/TPGS at 25:70:5(m/m) without the addition of HSA-PEHA and otherwise prepared by thesame method as LCAN. After 4 h at 37° C., the transfection medium wasremoved and cells were washed three times with PBS. Fresh medium wasthen added to the cells. At 48 h after the transfection, the cells wereharvested. Briefly, total RNA was extracted using Trizol reagent(Invitrogen) and cDNA was synthesized by incubating RNA with randomhexamer primer (Perkin Elmer, Boston, Mass.), and then with reversetranscriptase (Invitrogen), reaction buffer, dithiothreitol, dNTPs andRNAsin, followed by incubation at 42° C. for 60 minutes and 94° C. for 5minutes in a thermal cycler (Applied Biosystems, Foster City, Calif.).The resulting cDNA was amplified by real-time PCR (ABI Prism 7700Sequence Detection System, Applied Biosystems) using bcl-2 primers andprobes:

forward primer  [SEQ ID NO. 1] CCCTGTGGATGACTGAGTACCTG; reverse primer [SEQ ID NO. 2] CCAGCCTCCGTTATCCTGG;  and, probe  [SEQ ID NO. 3])CCGGCACCTGCACACCTGGA.

Housekeeping gene ABL mRNAs were also amplified concurrently and Bcl-2mRNA was normalized to ABL mRNA levels.

The results are displayed in FIG. 7. These results showed thatLCAN-G3139 was much more effective in Bcl-2 down regulation thanLN-G3139, a typical LN formulation of G3139 that does not containHSA-PEHA. These data showed that LCAN-G3139 is a superior composition tomost LNs and can be used to deliver antisense ASOs and otheroligonucleotide drugs, such as siRNA, miR mimics, and anti-miR oligos.

Example 4

Low molecular weight PEI(600) was used in this example. Alternative lowmolecular weight polymers, such as pentaethylenehexamine (PEHA), mayalso be conjugated using similar techniques.

HSA-PEI conjugates were produced using EDC. 42 mg HSA and 188 mg PEI(MW=600) (molar ratio HSA:PEI 1:500) were combined in HBS to a totalvolume of 2.0 mL in a small vial, adjusted to pH 8.0. Due to the highlyalkaline nature of PEI, a PEI stock solution was titrated to pH 8.0prior to mixing. EDC was allowed to equilibrate to room temperaturebefore adding 9.60 mg EDC (80 fold molar excess relative to HSA), slowlyto a stirring solution of HSA and PEI. The mixture was reacted for 1 hat room temperature with stirring. pH was maintained at ˜9.0 over thecourse of the reaction. The product was passed through a PD-10 column toremove unreacted reagents. Gel mobility shift analysis was conducted todetermine the DNA condensation efficiency of the HSA-PEI conjugate.

In an alternative conjugate method that introduces a reducible liner,HSA-PEI conjugates were produced using Traut's reagent. 42 mg HSA and188 mg PEI (MW=600) (molar ratio HSA:PEI 1:500) were combined in HBS toa total volume of 2.0 mL in a small vial, adjusted to pH 8.0. Due to thehighly alkaline nature of PEI, a PEI stock solution was titrated to pH8.0 prior to mixing. 3.5 mg Traut's reagent (40 fold molar excessrelative to HSA) was dissolved in 10 μL DMSO and added slowly to astiffing solution of HSA and PEI. The mixture was reacted for 2 h atroom temperature with stiffing. A pH of 8.0 was maintained over thecourse of the reaction. Disulfide crosslinking resulting in oxidation ofsulfhydryls led to the formation of an HSA-PEI conjugate. This productwas purified by passing through a PD-10 column to remove unreactedreagents. Gel mobility shift analysis was conducted to determine the DNAcondensation efficiency of the conjugate.

In yet another method of synthesis, HSA-PEI conjugates were producedusing SPDP. 42 mg HSA, activated by Traut's reagent, and 188 mg PEI(MW=600) activated by SPDP (molar ratio HSA:PEI 1:500) were combined inHBS to a total volume of 2.0 mL in a small vial, adjusted to pH 8.0.This resulted in the formation of albumin-PEI conjugate via disulfidelinkages. Gel mobility shift analysis was conducted to determine thecondensation efficiency of the conjugate.

Albumin-PEI conjugates were produced using DTBP. 42 mg HSA and 188 mgPEI (MW=600) (molar ratio HSA:PEI 1:500) were combined in HBS to a totalvolume of 2.0 mL in a small vial, adjusted to pH 8.0. Due to the highlyalkaline nature of PEI, a PEI stock solution was titrated to pH 8.0prior to mixing. 3.9 mg DTBP (20 fold molar excess relative to HSA) wasdissolved in 10 μL DMSO and added slowly to a stirring solution of HSAand PEI. The mixture was reacted overnight at room temperature withstirring. A pH of 8.0 was maintained over the course of the reaction andthe product was passed through a PD-10 column to remove unreactedreagents. Gel mobility shift analysis was conducted to determine thecondensation efficiency of the conjugate.

Example 5

LNs containing HSA-PEI conjugates were produced. HSA-PEI at various w/wratios (0, 0.5, 1, 3, 6:1, HSA:ODN w/w) were combined with ODN LOR-2501(0.2 μM) (purchased from Alpha DNA) to find the optimal retardationratio using gel mobility shift analysis. Retardation occurred at 3:1(HSA:ODN w/w) (FIG. 1). DDAB, CHOL, and TPGS lipid stocks dissolved in100% ethanol were combined at a molar ratio of 60:35:5. 100 μL lipidmixture in ethanol was added to 900 μL 1×PBS buffer as to form empty LNsin 10% ethanol. The HSA-PEI/ODN complex was then combined with the emptyLNs to form LCANs. The formulation was briefly vortexed and allowed tostand for 15 m at room temperature before transfection into KB cells.Zeta potential analysis was completed on the LCAN containing HSA-PEI andODN LOR-2501 (purchased from Alpha DNA) at the ratio LN:HSA:ODN=10:1, 2,3:1. The concentration of ODN used was 0.2 μM (FIG. 2). All LCANscontaining HSA-PEI exhibited a positive charge ranging between 5 and 25mV. LCANs without HSA-PEI were neutrally charged. FIG. 3 displays thedownregulation of RNR R1 mRNA expression by LOR-2501 in LCANs.

KB cells, grown in RPMI 1640 medium at 37° C. under 5% CO₂ atmosphere,were plated 24 h prior to transfection at a density of 3.0×10⁵ cells perwell in a 6-well plate. Cells were grown to approximately 80% confluencyand the serum-containing media was removed. Cells were transfected with1000 μL transfection media and treated for 4 h. Transfection occurred inthe presence of 0% and 10% serum-containing RPMI 1640 media. Experimentswere performed with 3 replicates. After treatment was completed, cellswere washed with 1×PBS and serum-containing RPMI 1640 was restored. At48 h after treatment was completed, cells were analyzed for RNR R1expression levels by RT-PCR with actin as a housekeeping gene. Resultsare shown in FIG. 2. Under serum-free conditions, the 1:3, ODN:HSA LCANformulation showed the greatest transfection efficacy. Conversely, in10% serum, the 1:1 ODN:HSA LCAN formulation was the most efficacious.Cell viability 48 h after treatment was assessed by MTT assay (FIG. 4).A similar experiment involving conjugation of PEHA-to-albumin wascompleted and showed similar transfection activity (FIGS. 7 and 8).

Example 6

LCAN for delivery of anti-miR-221 into CAL-51 breast cancer cells wasstudied.

LCAN (using HSA-PEI based APC) were prepared as described above. CAL-51(triple negative breast cancer) cells were plated 24 h prior totransfection in a 6-well plate at a density of 2×10⁴ cells/cm² inDMEM/F12 media supplemented with 1% penicillin/streptomycin and 10% FBS.LCAN was combined with anti-miR-221 (100 nM) to gauge its ability toupregulate the downstream targets of miR-221, p27/Kip1 and the estrogenreceptor alpha (ERα). CAL-52 cells were transfected in the presence of20% serum. Treatment was allowed to proceed for 4 h at which time thetransfection medium was removed and replaced with fresh media(supplemented with 10% FBS). Cells were allowed to proliferate for anadditional 44 h before the start of RT-PCR. RNA from cells was extractedwith TRIzol Reagent (Life Technologies) and cDNA was generated bySuperScript® III First-Strand Synthesis System (Life Technologies) perthe manufacturer's instructions. RT-PCR was then performed using SYBRgreen (Life Technologies) and primers for p27/kip1 (Alpha DNA) and ERα:

forward: [SEQ ID NO. 4] 5'CGGAGCACGGGGACGGGTATC-3′, reverse:[SEQ ID NO. 5]) 5'-AAGACGAAGGGGAAGACGCACATC-3′.

β-actin was used as a control. As demonstrated in FIG. 10 and FIG. 11,LCAN/anti-miR-221 led to moderate increases in p27/Kip1 expression andslight increases in ERα expression.

Example 7

HSA-PEHA conjugates were synthesized at a relatively large scale. TheHSA:PEHA:EDC molar ratio used during synthesis was 1:1500:200 (mol/mol).5 g PEHA (MW 232.37, technical grade) was dissolved in 80 mL of ddH₂Oand then adjusted to pH 8.0 using 1 M HCl. 1 g (4 mL) of HSA (25%,Octapharma)) and then 562.5 mg of1-ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC, dissolved in DMSO)were added into the PEHA solution under stirring. The reaction continuedfor 3 h at room temperature. The mixture was then dialyzed using MWCO10,000 Spectrum membrane against ddH₂O at 4° C. The buffer was replacedevery 3-4 h until amines from PEHA became undetectable by the standardninhydrin or TNBS amine essay in the external buffer at the 3 h timepoint at the end of the dialysis cycle. For further scaled-up synthesis,the dialysis procedure can be replaced by tangential flow diafiltration,e.g., using a Millipore Pellicon cassette system or a Spectroporhollowfiber system. This method can also be used to concentrate theproduct to a desirable concentration. The product can be passed througha 0.22 μm sterile filter into a sterile container and stored at 4° C.For long-term storage, the product can be stored at −20° C. The productcan also be lyophilized.

The product protein concentration was determined using BCA proteinassay. The amine content of the HSA-PEHA conjugate was determined byTNBS assay or MALDI-TOF MS based on change in molecular weight relativeto HSA. Gel permeation chromatography combined with amine TNBS assay isused to demonstrate the lack of crosslinked HSA and the absence of freePEHA in the product. Due to the modest cost of the reagents used, theyield of the reaction is not critical. The purity of the product isexpected to be very high. Exact product specifications can be definedbased on PEHA-to-HSA ratio and higher limits of crosslinked HSA and freePEHA in the final product.

Certain embodiments of the formulations and methods disclosed herein aredefined in the above examples. It should be understood that theseexamples, while indicating particular embodiments of the invention, aregiven by way of illustration only. From the above discussion and theseexamples, one skilled in the art can ascertain the essentialcharacteristics of this disclosure, and without departing from thespirit and scope thereof, can make various changes and modifications toadapt the compositions and methods described herein to various usagesand conditions. Various changes may be made and equivalents may besubstituted for elements thereof without departing from the essentialscope of the disclosure. In addition, many modifications may be made toadapt a particular situation or material to the teachings of thedisclosure without departing from the essential scope thereof.

What is claimed is:
 1. A lipid nanoparticle comprising, at least onelipid, and at least one albumin-polymer conjugate (APC), wherein thepolymer comprises at least one positively-charged polymer.
 2. The lipidnanoparticle of claim 1, wherein the APC comprises a hyper-cationizedalbumin-polycation conjugate.
 3. The lipid nanoparticle of claim 1,wherein the lipid nanoparticle has a zeta potential of about 0 to about+40 mV.
 4. The lipid nanoparticle of claim 1, wherein the APC is capableof binding at least one oligonucleotide.
 5. The lipid nanoparticle ofclaim 1, wherein the positively-charged polymer comprises a polyamineselected from the group consisting of: spermine, dispermine,trispermine, tetraspermine, oligospermine, thermine, spermidine,dispermidine, trispermidine, oligospermidine, putrescine, polylysine,polyarginine, a polyethylenimine of branched or linear type, andpolyallylamine.
 6. The lipid nanoparticle of claim 1, wherein the atleast one positively-charged polymer comprises a polyethylenimine. 7.The lipid nanoparticle of claim 6, wherein the polyethylenimine has amolecular weight not greater than 50 kDa, or has a molecular weight offrom about 200 to about 2000 Da.
 8. The lipid nanoparticle of claim 6,wherein the polyethylenimine is conjugated to human serum albumin. 9.The lipid nanoparticle of claim 1, wherein the at least onepositively-charged polymer comprises tetraethylenepentamine (TEPA). 10.The lipid nanoparticle of claim 1, wherein the positively-chargedpolymer comprises a mixture of two or more low molecular weightpolymers.
 11. The lipid nanoparticle of claim 1, wherein the albumin isconjugated to the at least one positively-charged polymer via one ormore cross linking agents.
 12. The lipid nanoparticle of claim 1,wherein the lipid nanoparticle comprises pentaethylenehexamine (PEHA)conjugated to human serum albumin (HSA).
 13. The lipid nanoparticle ofclaim 12, wherein an average of about eleven (11) PEHA molecules arelinked to each HSA molecule.
 14. The lipid nanoparticle of claim 1,wherein the at least one lipid comprises one or more of: a cationiclipid, a neutral lipid, a PEGylated lipid, and cholesterol.
 15. Thelipid nanoparticle of claim 1, wherein the at least one lipid comprises1,2-dioleoyl-3-trimethylammonium-propane (DOTAP),L-α-phosphatidylcholine (SPC), and d-alpha-tocopheryl polyethyleneglycol 1000 succinate (TPGS).
 16. The lipid nanoparticle of claim 15,wherein the lipids are in a 25:70:5 molar ratio of DOTAP:SPC:TPGS. 17.The lipid nanoparticle of claim 1, wherein the lipid nanoparticleencapsulates at least one molecule selected from nucleic acids,chemotherapeutic agents, and combinations thereof.
 18. The lipidnanoparticle of claim 17, wherein the encapsulated molecule comprises anucleic acid selected from plasmid DNAs, antisense oligonucleotides,miRs, anti-miRs, shRNAs, siRNAs, and combinations thereof.
 19. The lipidnanoparticle of claim 17, wherein the encapsulated molecule comprises atherapeutic agent selected from: antineoplastic agents, anti-infectiveagents, local anesthetics, anti-allergics, antianemics, angiogenesis,inhibitors, beta-adrenergic blockers, calcium channel antagonists,anti-hypertensive agents, anti-depressants, anti-convulsants,anti-bacterial, anti-fungal, anti-viral, anti-rheumatics,anthelminithics, antiparasitic agents, corticosteroids, hormones,hormone antagonists, immunomodulators, neurotransmitter antagonists,anti-diabetic agents, anti-epileptics, anti-hemmorhagics,anti-hypertonics, antiglaucoma agents, immunomodulatory cytokines,sedatives, chemokines, vitamins, toxins, narcotics, imaging agents, andcombinations thereof.
 20. The lipid nanoparticle of claim 17, whereinthe encapsulated molecule comprises a nucleic acid therapeutic agent.21. The lipid nanoparticle of claim 20, wherein the nucleic acidtherapeutic agent is selected from: pDNA, siRNA, miRNA, anti-miRNA, ASO,and combinations thereof.
 22. The lipid nanoparticle of claim 20,wherein the nucleic acid therapeutic agent is stabilized bymodifications to substituent NA base units, by phosphorothioatesubstitution of phosphodiester linkers, and/or by 2′-O-methylation ofthe ribose units.
 23. The lipid nanoparticle of claim 1, wherein thelipid nanoparticle has a diameter under about 300 nm
 24. The lipidnanoparticle of claim 1, wherein the lipid nanoparticle has a diameterunder about 200 nm.
 25. The lipid nanoparticle of claim 1, wherein thepolymer is bound to an external surface of the lipid via directconnection or via a linker.
 26. The lipid nanoparticle of claim 16,wherein the lipid nanoparticle has an encapsulation efficiency of themolecule of at least about 40% or higher.
 27. The lipid nanoparticle ofclaim 1, further including a polyethylene glycol-conjugated lipid. 28.The lipid nanoparticle of claim 27, wherein the polyethyleneglycol-conjugated lipid comprises one or more of: polysorbate 80, TPGSand mPEG-DSPE.
 29. The lipid nanoparticle of claim 27, wherein thepolyethylene glycol-conjugated lipid is present at a concentration lessthan about 15.0 molar percent.
 30. The lipid nanoparticle of claim 1,further comprising a ligand capable of binding to a target cell or atarget molecule on a cell surface.
 31. The lipid nanoparticle of claim30, wherein the ligand is an antibody or an antibody fragment.
 32. Thelipid nanoparticle of claim 30, wherein the ligand is selected fromcRGD, galatose-containing moieties, transferrin, folate, low densitylipoprotein, or epidermal growth factors.
 33. A pharmaceuticalcomposition comprising the lipid nanoparticle of claim 1 and apharmaceutically acceptable excipient.
 34. The pharmaceuticalcomposition of claim 33, wherein the pharmaceutical composition isadministered perorally, intravenously, intraperitoneally,subcutaneously, or transdermally.
 35. The pharmaceutical composition ofclaim 33, wherein the pharmaceutical composition is prepared as anorally administered tablet, an inhalant, or a suppository.
 36. Thepharmaceutical composition of claim 33, wherein the pharmaceuticalcomposition is prepared as a sterile solution, a sterile suspension, ora lyophilized powder.
 37. A method of making a lipid nanoparticle, themethod comprising: adding at least one lipid to a human serumalbumin-pentaethylenehexamine (HSA-PEHA) conjugate to form a mixture;adding at least one therapeutic molecule to the mixture; and subjectingthe mixture to a dialysis or diafiltration step to make a lipidnanoparticle.
 38. The method of claim 37, wherein the at least one lipidcomprises DOTAP, SPC, and TPGS at a 25:70:5 ratio.
 39. The method ofclaim 37, wherein the therapeutic molecule is selected from pDNAs,antisense oligonucleotides, miRs, anti-miRs, shRNAs, siRNAs, orcombinations thereof.
 40. The product made from the method of claim 39.41. A method treating a disorder, the method comprising administering aneffective amount of a pharmaceutical composition of claim 33 to asubject in need thereof.
 42. A delivery system comprising: a. at leastone lipid; and b. a macromolecule conjugated to a polymer; c. whereinthe macromolecule encapsulate a nucleic acid.
 43. A method of using alipid nanoparticle, comprising: a. encapsulating a nucleic acid in alipid nanoparticle, wherein the lipid nanoparticle comprises albuminconjugated to a polymer; b. incorporating the lipid nanoparticle into apharmaceutical composition; and c. administering the pharmaceuticalcomposition to a patient in need thereof.